The Effect of Cryopreservation Solutions and Cooling Rates on the Structural and Cellular Integrity of Frozen/Thawed Horse Testicular Tissue.

The ability to promote the growth of early-stage spermatogonial stem cells (SSCs) recovered from frozen tissues could lead to significant advances in breeding technology. This study is crucial in preserving the genomes of rare genotypes or species facing extinction. There is a need to develop methods to optimize available cryopreservation methods for the repopulation of endangered species. In this study, modeling and experimental approaches were used to investigate the interplay between freezing cooling rate and protectant solution for cryopreservation of testicular tissue from a two-year-old horse. Equids were selected for this study because they are a good model for the endangered Northern White Rhinoceroses being members of the same mammal order, Perissodactyla (Bigalke). The solutions of interest were Dulbecco's Modified Eagle's Medium (DMEM), and Phosphate Buffer Saline (PBS). Solutions were tested using three slow freeze cooling rates ad compared to fresh and refrigerated fixed tissues to deduce the best combination for cryopreservation. Comparisons were made based on: (1) structural changes including damage to the seminiferous tubules, epithelium detachment, and tubule perimeters or sizes, and (2) cellular changes indicated by cell density, and pyknosis, or cellular death. Data collection and statistical analysis indicated that freezing cooling brought about by decreasing the temperature to -7 ºC at a rate of a 2 ºC, followed by a five-minute hold -7 ºC, and a subsequence drop to -40 ºC at a rate of 0.3 ºC/min yielded the best results across all test parameters. Furthermore, PBS was shown to be superior to DMEM in all of the test parameters with the exception of cell density. This work contributes to the overall body of knowledge aimed at defining optimal conditions for the cryopreservation of testicular tissue and could have immense ramifications related to improving the integrity and quality of frozen/thawed gonadal material and improve efficacy when handling valuable samples from extinct species.